...For bacteria snipping apart DNA that bears certain signature sequence... Tools that snip apart DNA strands in defined locations are essentia...But this could change says Howard Hughes Medical Institute investig... We can direct it to any site we select says Doudna. Because th...

For bacteria, snipping apart DNA that bears certain signature sequences is a defense mechanism. For scientists working in the lab, the same strategy can be a powerful research tool. With a newly discovered component of an adaptive bacterial immune system, scientists have identified a targeted method of slicing DNA that they say can be easily customized for a variety of applications in the lab.

Tools that snip apart DNA strands in defined locations are essential for editing genomes in the laboratory to study or alter gene function. To target the specific site in the genome they are interested in, researchers often have to design and produce a protein that will recognize and bind to that particular DNA sequence, a laborious and time-consuming process.

But this could change, says Howard Hughes Medical Institute investigator Jennifer Doudna at the University of California, Berkeley. In a paper published online June 28, 2012, in the journal Science, she and her collaborator Emmanuelle Charpentier of the Laboratory for Molecular Infection Medicine at Ume University in Sweden report the discovery of an RNA-based complex used by bacteria to guide the DNA-cutting enzyme Cas9 to specific sites in the genomes of viruses and other invaders, thus silencing their genes. From this bacterial complex, Doudna and her colleagues have crafted a system with which an easily programmable guide RNA can be used in the lab to direct Cas9 to cleave double-stranded DNA at a desired target sequence.

"We can direct it to any site we select," says Doudna. "Because the guide RNA contains both the structure required for Cas9 binding and a separate guide sequence that can base pair with DNA, we can program Cas9 to cleave a specific site by simply changing the guide sequence. This system offers a straightforward way to cleave any desired site in a genome, which could be used to introduce new genetic information by coupling it to well-known cellular DNA recombination mechanisms."

Doudna, a biochemist, is interested in the molecular mechanisms by which RNA can influence gene expression. She says the discovery of an RNA-programmable DNA cleaving enzyme stems from a collaboration with Charpentier established last year. Both labs were studying different aspects of RNA-based defensive systems in bacteria that recognize and destroy the genomes of invading viruses and plasmids.

First described in the late 1980s, the system is called CRISPR, for Clustered Regularly Interspaced Short Palindromic Repeats. In response to a viral infection or plasmid transformation, bits of the invader's DNA known as proto-spacers are integrated into the host chromosome. The captured sequences are transcribed and processed to form short crRNAs, which serve as RNA recognition elements that bind to corresponding sequences in foreign DNA. Guided by the RNAs, proteins known as Cas (CRISPR-associated) then move in and attack the invaders, cleaving their DNA and silencing them.

Researchers studying CRISPR systems in various bacteria had found that in most cases a single crRNA joins with a large, multi-protein complex to attack viruses and plasmids. However, Charpentier had discovered that in Streptococcus pyogenes, a human pathogen, crRNAs could only be produced in the presence of a second RNA, which they called a trans-activating crRNA (tracrRNA). In addition, the S. pyogenes and related CRISPR systems require just a single protein, Cas9, for immunity to viruses targeted by crRNAs.

Doudna's lab worked with the Charpentier lab to investigate how Cas9 and crRNAs function in this bacterial immune system. Martin Jinek, an HHMI Research Specialist in Doudna's lab, succeeded in purifying the Cas9 protein. Krzysztof Chylinski, a graduate student in the Charpentier lab who is located at the Max F. Perutz Laboratories at the University of Vienna, used that sample to show that Cas9 needed both crRNA and tracrRNA to guide and execute its attack.

"We then decided to test whether we could link these two RNAs into a single, chimeric RNA molecule," Doudna says. Combining the elements of the crRNA and tracrRNA that were necessary for Cas binding and DNA target recognition into a single molecule would make the system easier to manipulate for laboratory use, she explains. It worked: the result was a DNA-cleaving enzyme that can be programmed with a single RNA molecule to cleave specific DNA sites.

The next steps, Doudna says, are to test the single-RNA construct along with Cas9 to find out whether the RNA-programmed enzyme works in the cells of eukaryotic organisms, such as worms, plants, and humans. If that is successful, she anticipates many practical applications of the tool. For biotechnology efforts ranging from engineering biofuel-producing microorganisms to enabling cell-based medical therapies, "having a simple and inexpensive tool for genome editing available will be very important," she says.

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